This invention relates to an improved method for producing heterologous proteins in insect cells, specifically in whole insect hosts.
Recent advances in the genetic engineering of baculoviruses have made possible the production of heterologous proteins in insect host cells grown in in vitro culture. One strategy which has achieved a certain measure of success involves (1) preparing a recombinant baculovirus which contains a DNA sequence encoding the heterologous protein operatively linked to the baculovirus polyhedrin promoter; (2) infecting cultured insect host cells with the recombinant virus; (3) growing the infected insect cells under suitable conditions for viral replication and protein expression and (4) recovering the desired heterologous protein thereby expressed from the insect cells or culture medium. Relatively high levels of expression of the desired protein can be obtained by the aforementioned strategy, in part because the polyhedrin promoter is such a strong promoter.
In considering the biology of this approach it should be kept in mind that baculoviruses are typically packaged in two forms: nucleocapsids may be occluded in the nucleus of infected cells in particles known as "polyhedral inclusion bodies" (PIBs) of which the predominant structural protein is polyhedrin or they may bud through the membrane of the infected cell thereby acquiring a membrane envelope to form non-occluded virus (NOV) particles. As a result of the usual deletion of all or part of the polyhedrin structural gene, or insertion of the heterologous gene within the polyhedrin gene locus involved in using the virus as an expression vector, the recombinant baculovirus used in the aforementioned approach is no longer capable of directing the synthesis of functional polyhedrin in infected cells. Horizontal transmission of the viral infection at the organismal level, i.e. from insect to insect, does not generally occur in the absence of the PIB form of the virus which cannot be produced without its principal structural protein, polyhedrin. Thus, infection with the recombinant virus results in the production of non-occluded viral progeny (NOVs) capable of spreading the infection from cell to cell within an infected insect or cell culture, but not from insect to insect.
Therefore, while the above-described recombinant baculovirus may be used to produce a heterologous protein in cultured insect cells or in a suitably inoculated individual insect host, production of the protein in whole insects in commercially significant amounts and/or horizontal transmission of the recombinant baculovirus would require the inoculation of a huge number of individual insects. Such extraordinary requirements of manpower, time and expense render such methods impracticable.
A new mixed viral system has now been discovered which permits for the first time the practical production of biologically and/or commercially significant amounts of a heterologous protein in whole insects and horizontal transmission of recombinant baculoviruses using a recombinant baculovirus which is itself defective with respect to polyhedrin production.